The purpose of this project is to determine the specificity of ionizing radiation mutagenesis, primarily in mammalian cells. Ionizing radiation is a known mutagen and carcinogen and exposure to this agent can occur in the environment, the work place, and during medical diagnosis and treatment. The determination of the specificity of ionizing radiation mutagenesis should provide important clues to the mechanism by which this agent produces mutations and cancer. The approach which we will take to determine mutational specificity will be to utilize shuttle vector plasmids. These plasmids contain sequences from both animal viruses and bacterial plasmids, and are capable of replication in both mammalian cells and bacteria. Many of the experiments planned for this project will utilize a mouse fibroblast cell line (A912) which contains a retroviral shuttle vector integrated into its chromosomal DNA. The shuttle vector contains the Escherichia coli gpt gene as a target for mutagenesis. Mutations will be induced in this gene using both X-rays and neutrons. The target gene will then be recovered from the mouse cells and the exact nature of the mutation will be determined by DNA sequencing. We will also determine mutational specificity following mutagenesis in the presence of agents or conditions which modulate the effects of ionizing radiation. These will include radioprotectors, anoxic conditions, and dose fractionation. We also plan to develop and utilize new systems to study mutational specificity in other types of cells. These will include: 1) a bacterial plasmid containing the gpt gene to study spontaneous and neutron-induced mutagenesis in bacteria; 2) a retroviral shuttle vector system to study mutational specificity in Chinese hamster cells; and 3) retroviral and Epstein-Barr virus shuttle vector systems to study ionizing radiation mutagenesis in radioresistant human tumor cells.